CAB Reviews 2019 14, No. 032

Gill diseases in marine with an emphasis on

Marco Rozas-Serri

Address: Laboratorio Pathovet Ltd, Newenko Group SpA, Palena 280, Puerto Montt 5480000, Chile. MR-S: 0000-0002-1693-7178.

Correspondence: Marco Rozas-Serri. Email: [email protected]

Received: 4 September 2018 Accepted: 8 April 2019 doi: 10.1079/PAVSNNR201914032

The electronic version of this article is the definitive one. It is located here: http://www.cabi.org/cabreviews

© CAB International 2019 (Online ISSN 1749-8848)

Abstract

Gill diseases are a growing health challenge in salmon farming worldwide, but many gaps remain in our knowledge. Gill diseases are generally complex and multifactorial disorders, often with presumable spatial and temporal distribution patterns, but are highly difficult to effectively prevent and control. The term complex gill disease (CGD) includes a wide range of clinical disease presentations on the gills of farmed salmon; usually, CGD presents from the end of summer to early winter. The pathogens involved include perurans, Tenacibaculum maritimum, Candidatus Piscichlamydia salmonis, Candidatus Branchiomonas cysticola, Desmozoon lepeophtherii (syn. Paranucleospora theridion) and viruses, such as the paramyxovirus (ASPV) and salmon gill poxvirus (SGPV). Amoebic gill disease (AGD) is perhaps the most significant disease in terms of gill health and economic impact. AGD results in high mortality, reduced production performance and impaired fish welfare. This review summarizes and analyses CGD research, outbreaks and treatment, with a focus on AGD, as well as on knowledge gaps and avenues for future research.

Keywords: Complex gill diseases, Salmon farming

Review Methodology: We searched the PubMed and CAB Abstracts databases (keyword search terms used: gill, infectious, diseases, salmon). In addition, we performed a general search of all keywords using EndNote X9 software (Clarivate Analytics, Boston, MA, USA).

Introduction The difficulty in establishing the cause of multifactorial gill disease has resulted in an inconsistent classification. The gill is a vital multifunctional organ that provides gas Amoebic gill disease (AGD), caused by Neoparamoeba exchange and assists in osmotic and ionic regulation and the perurans (syn. perurans), is perhaps the most excretion of nitrogenous waste. Gill disorders represent a significant disease in terms of gill health and economic significant challenge to salmon producers worldwide and impact [2–6], but several other pathogens are also are a cause of high direct and indirect losses [1]. The gills potentially associated with gill disease [7]. The term are mainly predisposed to pollutants, environmental ‘proliferative gill inflammation’ (PGI) has been used to changes and parasitic, bacterial and viral infections describe recurrent gill disease outbreaks that occur in because they are in direct contact with the environment. autumn in salmon farms in Norway [8, 9]. Similar Gill diseases are generally complex and multifactorial pathologies to PGI also have been described as occurring disorders, often with presumable spatial and temporal in Scotland and Ireland [10]. ‘Proliferative gill disease’ distribution patterns, but they are very difficult to (PGD) is used as a nonspecific term derived from the effectively prevent and control. Compromised gill function appearance of gross lesions in the salmon gill. In Scotland, can lead to significant economic losses due to poor food gill disease occurs during the same season as in Norway and conversion performance, direct mortalities and the cost of has been referred to as PGD due to the proliferative treatment. histological features and uncertain aetiology. Gill disease

http://www.cabi.org/cabreviews 2 CAB Reviews Table 1 Pathogens associated with gill disease in marine salmon aquaculture Pathology/ Pathogen Salmon syndrome Geographical distribution References Neoparamoeba Atlantic salmon, rainbow Amoebic gill Australia, Norway, Chile, [21] perurans trout and disease (AGD) Scotland, Ireland, USA and [22] Canada [23] [2] [4] [24, 25] [26] [27] Candidatus Atlantic salmon Epitheliocystis Norway and Ireland [28] Branchiomonas [29] cysticola Candidatus [30] Piscichlamydia salmonis Desmozoon Atlantic salmon Complex gill Norway, Scotland and Ireland [31] lepeophtherii disease (CGD) [32] Atlantic salmon [33] paramyxovirus (ASPV) [34, 35] Salmon gill poxvirus (SGPV) Tenacibaculum Atlantic salmon, rainbow Tenacibaculosis Australia, Norway, Chile, [12] maritimum trout, coho salmon and Scotland, Ireland, USA and [13] Canada [14] [15] presentation in Scotland is considered to be virtually of bacterial colonies or using molecular diagnostics synonymous with PGI in Norway, but with a less [16–18]. Transmission of the bacterium can be through pronounced inflammatory response and inconsistent vas- seawater or directly from host to host [16]; however, cular changes. Similar gill diseases have been reported in (e.g. Phialella quadrata and Pelagia noctiluca)mayact Ireland, Chile and Canada [11]. as a vector for this pathogen [19, 20]. Atlantic salmon have Complex gill disease (CGD) is the term currently used to been found to be particularly susceptible to tenacibaculosis, refer to this varied syndrome of probable multifactorial with fish age (juvenile) and temperature (above 15 °C) aetiology and variable histopathology, and the term being identified as risk factors [16]. encompasses the syndromes referred to as PGI or PGD Epithelial cysts in the gills of seawater-farmed Atlantic in previously published articles [11]. CGD includes a wide salmon are a common finding during gill disease out- range of clinical disease presentations on the gills of farmed breaks [30] and have been associated with various putative salmon, and these diseases usually appear from the end of pathogens, including the bacteria Candidatus Branchiomonas summer to early winter, and environmental insults from cysticola and Candidatus Piscichlamydia salmonis [28, 36]. and/or are frequently involved. Epitheliocystis is characterized by the development of inclusions/cysts in the branchial epithelium in addition to chloride cells [28, 29, 37] and has also been documented Pathogens Involved in CGD as occurring in skin epithelial cells [38]. The pathology in gills associated with epitheliocystis includes hyperplasia, Table 1 summarizes the main pathogens associated with gill lamellar fusion and focal necrosis of epithelial cells [30]. diseases in marine salmon aquaculture. Bacteria such as A number of bacterial agents are associated with Tenacibaculum maritimum are the causative agents of epitheliocystis in salmonids [39]; however, Candidatus tenacibaculosis, gill rot and gliding bacterial diseases B. cysticola has recently been identified as a potential [12–16], and associated gill lesions were first described in agent of epitheliocystis in marine-cultured Atlantic salmon chinook salmon tshawytscha [15]. Gill infec- [28]. A molecular study found Candidatus B. cysticola at a far tions due to tenacibaculosis tend to present in lethargic fish greater density in fish with large numbers of epithelial cysts; with an increased respiratory rate and increased mucus on in addition, in situ hybridization (ISH) allowed identification the gills, along with pale patches of necrosis [12–15]. of the agent within cysts, which indicated a potential role Preliminary diagnoses of symptomatic fish are carried out for the agent in gill disease [30]. Some studies have via a microscopic examination of affected tissue that shows indicated that epitheliocystis is merely coincidental, motile filamentous bacteria [12–15]. Further definitive whereas others have observed it during PGI outbreaks confirmation should be performed through the isolation with associated mortality [8, 9]. Candidatus P. salmonis has

http://www.cabi.org/cabreviews Marco Rozas-Serri 3 previously been associated with gill disease. However, the advances in molecular techniques, SGPV has been shown to role of Candidatus P. salmonis is still relatively unclear, be far more widely distributed than previously believed and particularly with respect to whether it is a primary or is often found in addition to a number of other pathogenic secondary pathogen [36]. agents [35], which further highlights the multifactorial A microsporidian parasite, Desmozoon lepeophtherii (syn. nature of gill disease. Paranucleospora theridion), has recently been described In Ireland, Downes et al. [43] observed that between 12 [31, 40]. D. lepeophtherii is believed to have a complex life and 16 weeks after seawater transfer, colonization of the cycle involving both salmonis and Atlantic gills by D. lepeophtherii, Candidatus B. cysticola, T. maritimum, salmon [31], although salmon have been found to be SGPV and N. perurans commenced, and by week 16 of infected with the microsporidian in the absence of lice [41]. marine production, each of the pathogens was detected. The true significance of this parasite as a gill pathogen is still D. lepeophtherii and Candidatus B. cysticola were by far the unclear as it is frequently the most prevalent agent detected most prevalent of the potential pathogens detected. in gill samples, even in gills with no reported pathologies T. maritimum was found to be significantly correlated with [7, 9]. However, it was present in fish with PGI at up to temperature, thereby showing distinct seasonality. SGPV 30 times the levels observed in unaffected fish in one was found to be highly sporadic and was detected in the study [9], with a four-fold increase in another [7]. first sampling point, suggesting a carryover from the In a case from Scotland, D. lepeophtherii was the apparent freshwater stage of production. The results of model causative agent of the gill disease outbreak recorded which indicated no clear effect between any of the pathogens, was associated with a distinct proliferative and necrotic but the models showed that the only variable that had a pathology [10]. D. lepeophtherii may encourage immune consistent effect on the histology score was N. perurans. suppression, thereby increasing the susceptibility of the However, noninfectious disorders due to harmful algae host as well as facilitating the proliferation of pathogens blooms (HABs) and other challenges also play roles in the already present in the fish [7, 31]. The densities of the mortalities attributable to CGD [43]. Several species of microsporidian appear to be influenced by environmental marine phytoplankton have been recorded to be associated conditions, with higher densities being recorded during with fish mortalities, including Karenia mikimotoi, which has periods with the highest temperatures [7, 41]. Further been implicated in Atlantic salmon mortalities in Ireland, work is required to fully understand the relationship Scotland and Norway [44–47]. A HAB of Pseudochattonella between the marine environment and potential gill cf. verruculosa during the 2016 austral summer (January– disease pathogens [10]. The role of other parasites during March) killed nearly 12% of Chilean salmon produced, CGD, such as Parvicapsula pseudobranchiola, Ichthyobodo causing the worst mass mortality of fish and shellfish ever spp. and Trichodina spp., may accentuate gill disease, but recorded in the coastal waters of western Patagonia [48]. these parasites appear to be secondary pathogens [11]. The algae bloom coincided with a strong El Niño event and Atlantic salmon paramyxovirus (ASPV) and salmonid gill the positive phase of the Southern Annular Mode that poxvirus (SGPV) have been identified as having some altered the atmospheric circulation in southern South association with gill disease in Atlantic salmon, but their America and the adjacent Pacific Ocean. These events led effect remains relatively unclear. In 1995, a previously to very dry conditions and allowed higher than normal solar undescribed virus belonging to the paramyxoviridae genus radiation to reach the surface. Using a time series of was isolated from the gills of Atlantic salmon suffering from atmospheric, hydrologic and oceanographic data, PGI and was named ASPV [32]. However, subsequent León-Muñoz et al. [48] showed that an increase in the infection trials failed to determine the pathology or surface water temperature and reduced freshwater input mortalities in disease-free salmon, but the virus was resulted in a weakening of the vertical stratification in the associated with two cases of mortality in salmon farms in fjords and sounds of this region, which allowed the Norway [32]. Further studies examining the multifactorial advection of more saline and nutrient-rich waters, ulti- aetiology of PGI found no evidence for the involvement of mately resulting in an active harmful in coastal ASPV [31]. southern Chile. During a number of outbreaks of PGI in Norway, a DNA Although the worldwide occurrence of severe HABs in virus, SGPV, was first observed to infect epithelial cells, recent decades suggests a connection with anthropogenic causing hypertrophy and the degeneration of the nucleus, in climate change [49], the causal link needs to be established addition to causing 20 and 80% mortality in freshwater and at the regional scale [50]. Temperature has been identified marine sites, respectively [32, 42]. During the outbreak in as the most important environmental factor shaping the the marine site, Neoparamoeba sp. was also present, which structure of ocean communities [51]. Although may have contributed to the mortality [31, 39, 42]. the occurrence of HABs is controlled by multiple The effect of SGPV appears to be greatest when processes, temperature is a central organizing factor that recorded during freshwater production, and when it determines the potential for HABs to occur [52]. The coincides with smoltification, significantly increased levels continuance of ocean warming through the twenty-first of mortality have been recorded as the infection particu- century will promote the intensification and redistribution larly affects the gills and chloride cells [34, 35]. With of HABs around the world [53].

http://www.cabi.org/cabreviews 4 CAB Reviews

Figure 1. CGDs in Atlantic salmon. (a) Note the presence of organisms (arrow) retained in mucin and cellular detritus in the gill interlamellar space (H&E, 100 μm). (b, c) The pathology in the gills associated with PGD includes hyperplasia, lamellar fusion (black asterisk), focal necrosis of epithelial cells and mucous cells hyperplasia (H&E, 100×). (d) Gill lesion from an AGD-affected fish. Epithelial gill hyperplasia with an attached (black arrow), secondary lamellae fusion (white asterisk) and presence of interlamellar vesicles (black asterisk) (H&E, 80 μm, Rozas et al. [55]). Magnification showing the details of the amoeba nucleus, which has an amphiphilic core surrounded by an irregular basophilic ring (black arrow), and parasomes appeared within the eosinophilic cytoplasm (black head arrow) (H&E, 20 μm). (e) Gill pathology associated with PGI included presence of fibrin and/or dead cells in the lamellar vessels (short filled arrow), hyperplasia of epithelial cells (black asterisk), death of cells (filled arrowhead), and inflammatory cells (long filled arrow) in the epithelium. Many epitheliocysts is present (short unfilled arrow) (H&E, 20 μm [9]).

Rozas et al. [54] officially monitored gill diseases under potential risks that net cleaning poses to fish welfare. commission by the National and Aquaculture However, in situ measurements of gill health before and Service (Sernapesca) during the summer algae bloom of after net cleaning conducted in the field are necessary to 2016. A histological study was conducted on 182 surviving validate these findings. Both laboratory and field research fish sampled from 60 seawater farms during and after the should examine situations in which salmon are subject to algal bloom (February–April). The histological lesions repeated exposure to cnidarian cleaning waste at realistic observed were grouped into cellular changes (degeneration intervals. and necrosis) (36.26%; 66/182 fish), growth disorders (epithelial hyperplasia, mucosal cell hyperplasia and eosi- nophilic granular hyperplasia) (67.58%; 123/182 fish) and Amoebic Gill Disease circulatory disorders (telangiectasia, thrombosis and haem- orrhage) (53.29%; 97/182 fish). AGD is caused by the parasitic amoeba N. perurans and Initially (February–March), most of the fish presented affects Atlantic salmon gills [21]. The disease was first acute circulatory and cellular changes and the presence of identified in the mid-1980s, when it infected salmonids microalgae (76.05%; 108/142 fish) (Figure 1), probably farmed in Washington State, USA and Tasmania, Australia associated with the algae bloom, whereas in the last [55]. AGD has also been reported in Chile, Canada, samples (April), lesions were observed in the process of Norway, Scotland, Faroe Islands and Ireland [2, 4, recovery; these lesions included chronic progressive lesions 21–27, 55] (Table 2). After 2010, the occurrence of associated with biological agents, and specifically, an AGD in farmed Atlantic salmon has significantly increased increase in the frequency of N. perurans (77.5%; 31/40 in the Northeast Atlantic, initially in Ireland and Scotland in fish) was observed [53]. 2011–2012 and later northwards to the Orkney Islands in Finally, Bloecher et al. [56] recently confirmed the Shetland, Norway and the Faroe Islands in 2012–2013 [6]. negative impact that the hydroid nematocyst Ectopleura In Norway, AGD was observed for the first time in larynx can have on salmon gill health and highlighted the association with health problems in farmed Atlantic salmon

http://www.cabi.org/cabreviews Marco Rozas-Serri 5 Table 2 Salmonid species in which N. perurans infections temperatures [2, 26]. The temporal and spatial distribution have been verified, either by ISH or by PCR (conventional or of AGD cases in Chile may be closely related to the real-time PCR). All verified N. perurans infections represent ubiquitous nature of N. perurans, low rainfall and high farmed fish diagnosed with AGD salinity [4, 24, 25]. The increased AGD outbreaks during Agent summer may not be solely due to increased thermal stress Host/Country detection References in fish but also to increased amoebae attachment at 15 °C, Atlantic salmon which causes an increased gill pathology [60]. Australia (Tasmania) ISH, PCR [21, 23] United States of America ISH, PCR [23, 57] Chile ISH, PCR [4, 24, 25] The agent Ireland ISH [22, 23] Scotland ISH [23, 58] Norway PCR [2] The distinguishing feature separating Paramoeba, Canada PCR [27] Neoparamoeba and Janickina from other species of Faroe Islands PCR A. K. Olsen amoeba is the presence of the endosymbionts or para- (personal somes from the family ; an exception is the communication) in [6] Paramoeba eilhardi, which sometimes lacks parasomes [61, 62]. When in motion, trophozoites of the genus Paramoeba Australia (Tasmania) ISH [23] and Neoparamoeba usually possess several dactylopodia. A Chile PCR [24, 25] comparative study completed by Dyková et al. [63] Norway PCR [26] acknowledged the importance of molecular characteriz- Chinook salmon New Zealand ISH [23] ation, as differentiation between amoebae at the morpho- Coho salmon logical level is almost impossible. Chile PCR [24, 25] Although recent molecular evidence suggests that the genera Neoparamoeba and Paramoeba are paraphyletic and could be synonymized [64]; however, doing so would be at four sites in the autumn of 2006 [2]. Bustos et al.[4] premature, as nuclear SSU rDNA is highly conserved and described AGD in Atlantic salmon farmed in Chile and insufficient by itself to formalize such a change [65, 66]. confirmed that N. perurans was the causal agent. Therefore, until more scaled amoebae are sequenced and During an AGD epizootic in Chile, rainfall lower than the genes other than SSU rDNA are investigated, evidence is 15-year average was recorded from May to November insufficient to change the currently used nomenclature 2007, which was believed to be the most likely environ- [67–69]. mental factor for the timing of the outbreak [4, 24, 25]. The The phylogeny of the amoebae associated with the prevalence of AGD in Atlantic salmon farms was 55.7% Chilean epizootic was examined using the 18S rRNA gene, (29/52 farms), and the epidemic curve was observed and these findings were compared with 18S rRNA gene between May 2007 and June 2008, closely related to low sequences from 46 isolates of Neoparamoeba and an rainfall and high salinity (>32‰) [4, 24, 25]. Fish weighing outgroup [4]. Phylogenetic analysis of the Chilean gene more than 300 g reared in the Los Lagos Region during (GQ407108) sequence found that it clustered with summer and autumn showed a 3.7 (P = 0.0004), 4.2 the Australian and Norwegian isolates (EU326494) with (P = 0.0178) and 6.2 (P = 0.0031) times greater risk of 98.4–99.2 and 99.6% similarity, respectively, which suggests being AGD positive, respectively [24]. The reduction in that N. perurans has a universal distribution [4]. Atlantic salmon biomass reared in Chile was closely related For many years, advancement of research into the to the infectious salmon anaemia outbreaks, which may aetiology of AGD was inhibited due to the inability to have considerably increased the infection pressure of culture the causative agent [5, 6]. However, Crosbie et al. N. perurans on rainbow trout (63.2%, 12/19 farms) and [70] completed the isolation and in vitro culture of coho salmon (90.9%, 10/11 farms) [25]. N. perurans, and they were therefore able to fulfil Koch’s The occurrence of AGD in Ireland over the 2011/2012 postulates. The culture was maintained using malt yeast agar period presented some unique challenges for the Irish with seawater overlaid and subcultured every 3–4 days, from salmon industry, in particular, a shortage of well boats for which a clonal culture was established. After 70 days in treating infected fish and permission for the use of water culture, a clone successfully infected Atlantic salmon, sources by local authorities. Farms in Tasmania, which are causing AGD, which was subsequently reisolated and located at sites with a strong influence of fresh water due to confirmed by polymerase chain reaction (PCR) and ISH [70]. high levels of rainfall or with a strong freshwater input, are less impacted by AGD [59]. However, AGD has also been observed in farms in Tasmania at temperatures of 10.6 °C Risk factors and salinity of 7.2 ppt [60]. Outbreaks of AGD reported in Norway and Scotland Amphizoic marine amoebae are believed to be ubiquitous were described as being associated with higher water in the environment, whereas the role of reservoir

http://www.cabi.org/cabreviews 6 CAB Reviews populations, a possible mechanism of transmission to and the potential impacts on AGD of alternative strategies among farmed fish for many disease-causing amoebae, has against sea lice, such as ‘snorkel cages’ and the use of lights not been fully elucidated [57, 71]. Amoebae that can cause [83, 84], which encourage fish to spend more time in parasitic infections in farmed fish are known to be deeper waters, should be investigated. Recently, Wright free-living in the environment and may alter their life et al. [74] showed that snorkel technology has a place in the strategies given the correct circumstances [72]. toolkit of commercial salmon sea-cage farmers comanaging Such infections are generally poorly understood but may salmon lice and AGD outbreaks. occur due to adverse impacts on hosts from environmental When amoebae are isolated from a natural environment, stress factors, in particular, elevated temperatures, salinity they invariably bring bacteria to the culture [85], even when or the initial insult from zooplankton, which can leave the they are surface disinfected. Although many Neoparamoeba gills susceptible to infection [11]. N. perurans is transmitted isolates do not appear to be bacterivorous, if they are from fish to fish through the water, but information subcultured on a regular basis, bacteria are always present, regarding the dynamics of this spread is lacking. based on transmission electron microscopy [63], and in Water, biofouling, sediments and salmon parasites have some isolates taken from sediment, bacteria are present been tested for the presence of N. perurans to determine and multiply inside the amoebae. where the amoebae are located when they are not parasitic Although paramoebae feed on bacteria, their relation- [69]. Concentrations of N. perurans were found to be low in ship with bacteria may be more complex than just as a seawater from salmon farms in Tasmania [73–75] and source of nutrition. Similar to other phagotrophic eukar- Norway [76], even during AGD outbreaks, with maximum yotes, Neoparamoeba and Paramoeba are exposed to concentrations of up to 62.3 amoebae/l [73], which is much foreign DNA, which provides opportunities for horizontal lower than the concentration used in challenge trials in the gene transfer [86]. Currently, no evidence exists to support laboratory [57, 77]. The only time that any difference was the hypothesis that bacterial infection or the presence of observed in the abundance of N. perurans was during an nonpathogenic bacteria contributes to the severity of AGD AGD outbreak in early autumn (2014), when N. perurans [69]. This lack of evidence does not, however, mean that was more common in surface waters than at other depths bacteria are not somehow involved. Egan and Gardiner [87] [73]. The abundance of N. perurans in the water column was recently suggested that many diseases in the marine affected by changes in salinity following rainfall, where environment are the result of a microbiome disturbance amoebae were present only at higher salinities in deeper or microbial dysbiosis. The possibility that some or all waters and no obvious relationship existed between the paramoebiases are caused by imbalances in the microbiome depth distribution of N. perurans and Atlantic salmon [74]. of the afflicted should therefore not be ignored. There is no scientific evidence that sea lice have a While fish microbiome research is progressing, the focus significant role in spreading N. perurans. However, while has been on the gut microbiome, with the 16S rRNA gene N. perurans was recovered from sea lice in the USA [57], it being used to explore bacterial diversity [88]; little is known was not recovered in Norway. During an AGD epizootic about the microbiome of the gill. AGD can clearly be outbreak in Chile, exceptionally high levels of coinfection caused by exposure to N. perurans; however, neither the with rogercresseyi may have contributed to the potential for coinfection nor the possibility that microbial observed mortality [4]. A heavy infestation of salmon lice dysbiosis contributes to disease susceptibility can be may influence a case of AGD by increasing the burden on dismissed [87]. an already weakened fish [4]. Environmental factors, such as temperature, and long- Paramoeba spp. have been detected in the gills of term trends, such as climate change, in particular, may also wild couta, Thyrsites atun, caught in the vicinity of play a role in the increased effects of AGD on the salmon Atlantic salmon farms [76]. As greater emphasis is placed industry. In most geographical locations, this disease was throughout the industry on the reduction of medicinal first reported when temperature was above average [6]. treatments for sea lice, a renewed interest in these cleaner Climate change has been suggested to serve as a trigger for fish as biological controls has emerged [77, 78]. The microbial dysbiosis in the marine environment [87]. identification of N. perurans on the gills of cleaner fish Increasing temperatures or other stressful environmental species such as lumpsuckers (Cyclopterus lumpus) and conditions will serve to stress marine hosts, thereby wrasse (Labrus bergylta) is a major concern to the industry, impairing the immune responses of the hosts, which can as these cleaner fish may act as potential reservoirs or indirectly affect the composition of the host microbiome asymptomatic carriers and can infect to naive Atlantic and trigger an imbalance that leads to a disease outbreak salmon [76, 79, 80]. [87]. Microbial dysbiosis can also occur when environ- The pathogenesis of reinfection in the post-treatment mental factors directly affect an animal microbiome. period has been found to be identical to that of the initial Chalmers et al. [89] suggested that ploidy does not affect infection, although the source of the reinfection was not the manifestation or severity of AGD pathology or the identified [81]. Potentially, the treated salmon themselves serum innate immune response. Additionally, the serum may be the main source of reinfection, as some amoebae immune response of diploid and triploid Atlantic salmon remain on the gills following treatment [82]. Additionally, may not be significantly affected by AGD.

http://www.cabi.org/cabreviews Marco Rozas-Serri 7 Pathology the gills do not always coincide with AGD in salmon and are less reliable in the early stages of an infection [23, 24]. AGD clinically manifests as lethargy, anorexia, congregation Histology has been one of the primary methods for at the water surface and an increased ventilation rate identifying and diagnosing the causal agent, and it has also [59, 60]. Preliminary diagnosis of the infection is often done been utilized in the investigation of the host response through scoring of the white mucoid patches present on [60, 81]. Mitchell et al. [96] developed a histopathological the gills of infected fish [58, 75, 90], and these scores have gill scoring method that assigned a score of 0–3 for each been shown to be good indicators of AGD when the parameter associated with changes in gill health, including checks are performed by an experienced examiner [24, 60, lamellar oedema, lamellar hyperplasia, lamellar fusion and 75, 81, 91]. circular anomalies (necrosis and sloughing). The pathology of AGD has been well defined and is Rozas et al. [24] showed a moderate concordance level characterized by localized host tissue responses, including (k = 0.5319) between gross pathology and histology. The epithelial oedema, hyperplasia of the epithelial cells as well sensitivity and specificity of the gross pathology was 77.91 as mucous cells, fusion of lamellae and the development of and 71.05%, respectively. Although gross and histological interlamellar vesicles [43] (Figure 1). Amoebae may also screenings have provided valuable tools to the industry for attach in the histological examination, and these amoebae the regulation of AGD, they are still limited in their capacity should contain at least one Perkinsiella amoebae-like to identify the infectious agent [60, 81, 91, 96]. organism [2, 4, 21, 23–25, 60, 81, 91] (Figure 1), which is A number of laboratory techniques have been developed considered to be case defining [23–25, 60]. to confirm AGD in presumptively diagnosed fish [97–99]. By transmission electron microscopy, Wiik-Nielsen et al. Following the identification of N. perurans, Young et al. [97] [92] observed enlarged swellings in affected gill filaments developed a PCR assay that amplified a 636-bp region of the with fusion among adjacent lamellae, in addition to spherical 18S rRNA gene. Further investigation allowed for the amoebae, which appeared to be embedded within the development of ISH using oligonucleotides that bind to the epithelium and that subsequently left indentations with 18S rRNA gene, and this technique was utilized to confirm visible fenestrations. These fenestrated structures appeared that N. perurans was the predominant aetiological agent of to correspond with the presence of pseudopodia, which AGD in Tasmania, despite other amoebae species being were observed to penetrate the epithelium. previously associated with the disease [97]. The 18S rRNA Concentrations of amoeba from 10 to 500 amoeba/l have gene is generally chosen due to its high copy number, which been documented as causing AGD in naïve Atlantic allows for high sensitivity, and the 18S rRNA gene is an salmon, with the pathology observed in both gross and established marker for microbial identification, with a histological examinations appearing to be proportional to database of species-specific sequences [75]. This assay the concentration of amoeba initially used [93]. Differences was found to be specific and highly sensitive for the in virulence between the amoebae extracted from detection of N. perurans in gill samples and isolates of AGD-infected fish and amoebae cultured in vitro have noncultured gill-derived amoebae. been recorded throughout research into AGD, and ideally, Bridle et al. [75] developed and validated a real-time PCR studies should be conducted using well-characterized assay using SYBR® Green chemistry and an iQ5 Real-Time strains of N. perurans. Some evidence exists to show that PCR detection system (Bio-Rad NSW, Australia). The cultures maintained in a lab for extended periods of time primers used in this assay amplified a 146-bp portion of have displayed differences in virulence based on gill score the 18S rRNA gene from base 677 to 822 of N. perurans. [94]. Furthermore, the clonal strain of N. perurans originally The correlation between the PCR results of gill swabs taken used to fulfil Koch’s postulates [70] was found to have lost from infected salmon and gross gill scores showed potential virulence after 3 years in culture [95]. for the development of a nondestructive sampling regime for the detection of AGD [75]. Rozas et al. [24] developed a PCR assay to amplify the Diagnosis N. perurans 18S rRNA gene from gill clinical samples of AGD-affected fish. The oligonucleotides designed and used The most financially viable nondestructive means for for the detection of N. perurans showed amplicons of the diagnosing AGD at the commercial scale is through gross expected size (462 bp) from all analysed fish. This PCR was pathological assessment [24, 59, 60, 81] using various able to detect N. perurans genetic material in the gills of fish gill-scoring methods developed by Taylor et al. [3]. Tools with gross pathology and histological lesions characteristic such as gill scoring can be used to determine the severity of of AGD. High concordance (k = 0.95) between the PCR the AGD infection and the frequency of treatment [3, 5, 60, results and the histological examination was observed. 81, 91]. However, this approach is a presumptive means by A quantitative duplex real-time TaqMan®-based PCR was which to confirm the presence of AGD and is open to developed for the detection of N. perurans in Atlantic misinterpretation. The detection of lesions and patches salmon and rainbow trout, with a set of primers and probes only indicates an altered gill condition but lacks the ability to being used to amplify a 139-bp fragment specific to the identify the causative agent [60, 81]. Lesions and patches on N. perurans 18S rRNA gene [100]. Although the differences

http://www.cabi.org/cabreviews 8 CAB Reviews observed by Fringuelli et al. [100] between the parasite load response in the gills of AGD-affected Atlantic salmon at 10 and AGD score were not statistically significant, the gill days postinfection [105]. Contrary to previous studies, histopathology that was microscopically observed was not Pennachi et al. [105] suggested that N. perurans elicits a always associated with the presence of amoebae. This result, classical inflammatory response in the gills of AGD-affected together with the lack of agreement between the results fish and indicates that the mRNA expression of immune obtained by PCR and the gill histopathology examination, is genes within gill lesions misrepresents the cellular immune consistent with the histopathology of some of the samples response in the gills during AGD. The results obtained having been caused by other pathogens, waterborne during this study, most notably the increased expression irritants or a combination of these factors [100]. of TCR mRNA, which was strongly correlated with CD8 Treatments are triggered when moribund fish or fish mRNA, suggest the infiltration of T-cells and highlight the with advanced clinical signs of disease are sampled [99]. A importance of CD8+ T-cells and the possible involvement diagnostic method that allows for the early identification of of gill intraepithelial lymphoid tissue (ILT) (Table 3). the aetiological agent is essential, particularly as the cost of Fish affected by AGD show an increased length in the ILT treatment is highly demanding [96]. Fish with no obvious 28 days postexposure in the dorsal area of the gill arch, with pathology, either gross or histological, have previously a peak in lymphocyte density 7 days postexposure [108]. tested positive via PCR when sampled using a gill swab, Benedicenti et al. [106] examined the immune response suggesting that once correctly optimized, a PCR assay could involvement in AGD using Atlantic salmon post-smolts potentially be more sensitive than traditional diagnostic sampled 3 weeks after exposure to either 500 or methods [97]. 5000 cells/l N. perurans. Gene expression analysis was performed on the first gill arch including the ILT. The Th1, Th17 and Treg pathways were found to be significantly Immune response downregulated, mainly in samples from fish given the higher dose. By contrast, the Th2 pathway was found to be Early studies on the transcriptional responses to AGD have significantly upregulated by both infection doses. These shown no differences in the gill tissue expression of tumour results seen during late stage AGD suggest that either an necrosis factor (TNF)-α1, TNF-α2, interleukin (IL)-1β, immune evasion strategy, similar to the responses driven by inducible nitric oxide synthase and interferon (IFN)-γ helminthic parasites to avoid cell-mediated killing mechan- mRNAs compared with that of tissue from healthy fish isms, or an allergic reaction caused by the parasite is during the early onset of the disease in Atlantic salmon occurring (Table 4). [101]. With the progression of the disease, the IL-1β Valdenegro et al. [107] identified 186 and 322 non- mRNA level was found to be upregulated and lesion- redundant proteins in gill and skin mucus, respectively, restricted [102]. In AGD-affected tissue, significant down- based on stringent filtration criteria, and statistical analysis regulation of the major histocompatibility complex (MHC) demonstrated that 52 gill and 42 skin mucus proteins were class I (MHC-I) pathway-related genes occurred during the differentially expressed in mucus samples from later stages of infection and appeared to be mediated by the AGD-affected fish. By generating protein–protein inter- downregulation of IFN regulatory factor (IRF)-1, indepen- action networks, some of these proteins formed part of the dent of type I IFN, IFN-γ and IRF-2 expression [103]. cell-to-cell signalling and inflammation pathways, such as Within this condition, suppression of the MHC-I and C-reactive protein, apolipoprotein 1, granulin, cathepsin possibly the MHC-II pathways may inhibit the development and angiogenin-1. In addition, Marcos-López et al. [110] of acquired immunity and could explain the unusually high showed the upregulation of prohibitin, cyclophilin A, susceptibility of Atlantic salmon to AGD. apolipoprotein A1, ictacalcin, RhoGDP dissociation inhibi- However, anterior gradient-2 (AG-2), which is involved tor α, components of the heat shock proteins 70 family and in inhibiting the tumour suppressor protein p53 (p53) and histones H3a and H4 and downregulation of required for mucin (MUC) 2 post-transcriptional synthesis peroxiredoxin-5 and cofilin. Among the protein functions and secretion, was upregulated in AGD-affected gill tissue, identified were cell cycle regulation, cytoskeletal regulation, whereas p53 tumour suppressor protein mRNA was oxidative metabolism and immunity. concurrently downregulated in AGD lesions, suggesting a After four successive AGD challenges, no significant role for AG-2 and p53 in AGD pathogenesis [104]. MHC differences in the plasma or skin mucus levels of IgM were class II+ cells, considered to be antigen-presenting cells, observed between AGD-naïve and challenged fish, but IgM were found within gill lesions by immunohistochemistry, gene expression in gill lesions of AGD-affected fish and these cells exhibited variable levels of expression [104]. increased by up to 31 days after infection [109], which is A recent study has shown that the mRNA expression possibly explained by weak correlations between the levels of proinflammatory cytokines (IL-1β,TNF-α), cel- protein and mRNA abundances in cells and tissues. After lular markers of cell-mediated immunity (T cell receptor a single infection, Valdenegro et al. [109] showed that the (TCR)-α chain, cluster of differentiation (CD) 4, CD8, levels of serum or skin mucus IgM antibodies were not MHC-I, MHC-IIα), and antibody-mediated immunity affected, and no changes in the IgM or IgT transcription (IgM, IgT) are correlated with a classical inflammatory were induced.

http://www.cabi.org/cabreviews Marco Rozas-Serri 9 Table 3 Immune gene expression in the gills of Atlantic salmon during an AGD experimental challenge Infection stage Results Interpretation Ref. Early stage MUC-2, AG-2 upregulation AG-2 and p53 could have important roles in AGD [104] p53 downregulation pathogenesis Early stage TNFα, IL-1β,TCRα, CD4, CD8, N. perurans elicits a classical inflammatory response in the [105] MHC-I, MHC-IIα, IgM, IgT gills of AGD-affected fish upregulation Early stage TNFα, IL-1β, COX-2 upregulation N. perurans induces an early proinflammatory response in [101] the gills of AGD-affected fish Early stage Th1, Th17, Treg downregulation N. perurans promotes an allergic reaction and an [106] Th2 upregulation immune-evasion strategy to avoid cell-mediated killing mechanisms Late stage IgM upregulation N. perurans induces a humoral response in the gills of [107] AGD-affected fish Late stage MHC-I, MHC-II, IRF-1, IRF-2 This condition promotes the proinflammatory response, but [103, 104] downregulation the suppression of the MHC I and MHC II pathways could IL-1β, IL-8, COX-2 upregulation inhibit the development of acquired immunity Late stage IL-1β upregulation The proinflammatory response is maintained in late stages [102] of the AGD infection

Table 4 Activity of proteins related to the immune response and oxidative stress in Atlantic salmon affected with AGD under experimental and field conditions Day post-infection Challenge Results Interpretation Ref. Serum/gill-skin Experimental C-reactive protein, apolipoprotein 1, N. perurans modifies the cell cycle [107, 109] mucus granulin, cathepsin, angiogenin-1 regulation, cytoskeletal regulation, upregulation. oxidative metabolism and immunity. Levels of serum or mucus IgM antibody were not affected. Gills Experimental Prohibitin, cyclophilin A, apolipoprotein N. perurans modifies biological [110] A1, ictacalcin, RhoGDP dissociation processes such as cell signalling inhibitor α, HSP70, H3a, H4 and inflammation pathways. upregulation Peroxiredoxin-5 and cofilin downregulation. Gill mucus Field Decreased activity for peroxidase, Results obtained highlight the [111] lysozyme, esterase, protease and low capacity of gills to elicit a local IgM levels. response to the infection, indicate an impaired immune response at the later stages of the disease. Gills Field HAA and CATactivity decreased, The oxidative stress and impaired [112] whereas GR activity increased. antioxidant defences could contribute to the pathogenesis of late-stage AGD.

In the gill mucus, the IgM levels and activities of determined in the gills obtained from a natural peroxidase, lysozyme, esterase and protease decreased in AGD-infection at gill scores of (GS) 0 and GS 2 and after fish that had a greater time of exposure to infection by a post-freshwater treatment, and separate samples com- N. perurans and greater severity of the disease as well as a prising gill areas without and with lesions were taken [112]. sequential increase after treatment [111]. These results HAA analysis revealed a significant depletion of the demonstrate the ability of the gills to elicit a local response antioxidant capacity in infected gills and a recovery to to the infection and indicate an impaired immune response previous antioxidant levels after freshwater treatment. at the later stages of the disease, followed by a partial SOD activity did not differ between the lesion and reestablishment of the host immune status after treatment lesion-free areas, and CAT activity was diminished in the with fresh water. lesion areas of both infected and treated fish, whereas the Hydrophilic antioxidant activity (HAA) and antioxidant GR activity at GS 2 was increased in lesion areas relative to enzyme activity, including superoxide dismutase (SOD), lesion-free areas [112]. Oxidative stress and impaired catalase (CAT) and glutathione reductase (GR), were antioxidant defences could contribute to the pathogenesis

http://www.cabi.org/cabreviews 10 CAB Reviews of late-stage AGD, although knowledge gaps still exist, and Recently, Wright et al. [120] have shown that targeting this area requires further investigation. cell detachment rather than cell death with repeated freshwater treatments of shorter duration than typical baths could be used in AGD management. Thus, Treatment and control AGD-affected Atlantic salmon subjected daily to 30 min (sublethal) and 120 min (lethal) freshwater treatments for Freshwater bathing has been the standard method of 6 days consistently reduced the N. perurans cell numbers on treating the disease in Tasmania but is limited by access gills compared with daily 3 min freshwater or seawater to freshwater [6]. In cooler production areas, hydrogen treatments for 6 days. peroxide is an effective treatment, but the treatment is During the emergence of AGD in Australia, research recognized as having a narrow safety margin at higher focused on establishing an alternative chemotherapeutic temperatures [70] or where fish are compromised by agent; however, much of this research was relatively advanced AGD [113]. Affected are treated with unsuccessful [6]. A commonly used treatment in the freshwater bathing, a practice that costs the Australian aquaculture industry is hydrogen peroxide, which is utilized salmon industry A$41 million/year and adds A$1.12 per kg in the treatment of many external parasites and gill infections to production costs [114]. Some estimates have put the as well as fungal, bacterial and protozoan infections, including cost of AGD-related mortality between $12.55 million in infestations [121–123]. Farms in Ireland, Scotland Norway and $81 million in Scotland [114]. and Chile have good experience with using hydrogen Although significant research has been conducted on peroxide for the treatment of sea lice and had some treatments since AGD was first recorded, freshwater success in treating cases of AGD in 2011 and 2012 at bathing remains one of the most effective and essential dosage levels between 1000 and 1400 mg/l for 18–22 min. methods for the removal of the majority of amoebae that However, a major disadvantage of hydrogen peroxide for cause AGD [6, 71, 82, 115]. Current treatment strategies in the treatment of AGD is that a narrow safety margin exists, Australia involve monitoring of gross gill lesions and and at temperatures >13.5 °C, its use becomes hazardous prophylactic freshwater baths [3]. Reinfection of the gills [58, 122]. Moreover, hydrogen peroxide is routinely used can occur relatively quickly, varying from 1 to 2 weeks as a treatment for AGD and sea lice in Atlantic salmon post-freshwater bath, and increase in severity within 4 aquaculture, but gills showing decreased antioxidant weeks [60, 81, 82]. Since the initial outbreaks in Australia in capacity may be more susceptible to hydrogen peroxide the 1980s, farms have seen a requirement for an increase in damage or toxicity [112]. A mortality of 6.5–7.1% was the frequency of treatments, with some fish being treated recorded during in vivo trials with a concentration of up to 15 times a year [81, 82, 115, 116]. 1250 mg/l at 12 and 18 °C, which would be considered The mechanism by which freshwater bathing treats AGD commercially unacceptable [71]. The effects of hydrogen is by the osmotic effect, removing excess mucus and peroxide on Atlantic salmon gills were investigated in associated amoebae, thereby promoting healing of the gills relation to sea louse treatments, and it was determined [81, 82]. Findlay et al. [116] considered a number of factors, that exposure to 2.58 g/l for 20 min causes complete such as interactions among immune responses, health of mortality [124]. the fish and the gills, number of amoebae remaining Fallowing of sites and cage rotation have been identified following treatment, and environmental variables to be as having an effect on AGD, with fewer freshwater baths important relative to reinfection with AGD. being required and increased growth rates observed where Treatments are generally triggered when farms observe management practices were adjusted [125]. The current 30–40% of fish with gill scores of 2 or above [58]. Although experience suggests that the development of a vaccine freshwater bathing is effective at significantly reducing the against this disease remains a significant challenge for the amoeba gill load, with an 86 ± 9.1% reduction in the near future. Despite evidence for immunosuppression in number of live amoebae observed, the remaining the later stages of infection, experimental attempts to boost amoebae could potentially cause a reinfection within one the immune response have not been successful [5]. To date, week [82]. Water hardness has a noticeable effect on the immunostimulants or experimental vaccines have had little efficacy of freshwater bathing, with soft freshwater effect on the survival of AGD affected fish [5]. However, an (19.3–37.4 mg/l CaCO3) proving to be more effective at injection of CpG oligonucleotides and two experimental reducing the numbers of viable amoebae (73.9–40.9% of diets containing immunostimulants have produced some total count) [117]. encouraging results. Possibly, the dose and timing of the The physiological effects on salmon of freshwater bathing applications tested were not optimal, and optimization have also been investigated, and as a treatment, freshwater could lead to the development of an effective immunosti- bathing poses very little risk of side effects [118]. However, mulant [5]. Recently, Cano et al. [126] have shown that in any form of bathing treatment can be problematic as it vitro models can prove to be a promising tool to study host requires the fish to be confined by tarpaulin or cage skirt or responses to amoebae and may therefore reduce the be transferred to a well boat, which imposes a handling requirement for in vivo studies when evaluating alternative effect, causing acute stress to the fish [119]. therapeutants to AGD control.

http://www.cabi.org/cabreviews Marco Rozas-Serri 11 Breeding for disease resistance can contribute to the pathogen and disease is essential to reduce the costs of prevention and control of AGD, providing long-term AGD and ensure the sustainability of the salmon industry. cumulative benefits in selected stocks [125]. Robledo Further investigations into the potential for sea lice as a et al. [127] showed that resistance to AGD is a suitable vector for AGD would be important to fully assess if and trait for genomic selection, and the addition of this trait to how great a risk factor a heavy sea louse infestation would Atlantic salmon breeding programmes can lead to more be. Due to the complexity of the disease agents that resistant stocks. Gill damage and the amoebic load are potentially present on salmon farms at any time, an heritable (h2 ∼ 0.25–0.30) and show high positive corre- examination of hydroids is important in the context of lations, indicating they may be good measurements of host multifactorial diseases and as potential reservoirs and resistance to AGD. Although the genetic architecture of subsequent carriers of disease agents. At the same time, resistance appears to be largely polygenic in nature, two further studying the relationship between bacteria and regions on chromosome 18 are suggestive of an association AGD, as well as the importance of microbial dysbiosis, is of with both AGD-resistance traits. interest [69]. To design effective control strategies for gill disease in salmon aquaculture, the promotion of public–private cooperation between all of the entities involved in the Conclusions and Future Research salmon farming industry at national and international levels is fundamental, with the purpose of sharing information and Increasingly, CGD is a health challenge in salmon farming knowledge to ensure sustainability in the world salmon worldwide, but many gaps remain in our knowledge about industry. them. Coinfections are not uncommon on farms and may have synergistic effects on the host or reduce the effects of immunomodulation or treatment. However, a lack of Acknowledgements understanding exists regarding the interrelationships among all the pathogens involved in gill pathologies. We thank Marcela Lara, Osvaldo Sandoval and Álvaro These interactions should be explored using biological Gaete from the National Fisheries and Aquaculture Service, models for concomitant infections, and studies on the Sernapesca, Chile. In addition, we thank Ricardo Ildefonso, cohabitation of single pathogen-infected fish with naïve fish Arturo Riquelme, Soraya Barrientos and Victoria Jaramillo are required. from Pathovet Laboratory. To assess the risk of future outbreaks, we must develop a better understanding of the biology of the pathogens involved, e.g. the relationship between N. perurans loads in seawater and environmental variables, such as salinity, References temperature, bacterial load, turbidity and dissolved inor- 1. Rodger HD. Gill disorders: an emerging problem for farmed ganic nutrients. Studies on the interaction of the different Atlantic salmon ( salar) in the marine environment? 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